Cryptococcus neoformans is an opportunistic fungal pathogen, distinguished by a complex polysaccharide capsule required for virulence. The broad, long-term objectives of this work are to better understand the upstream metabolic events required for capsule synthesis in C. neoformans. The specific aims are directed at understanding the steps that provide the nucleotide sugar precursors essential for construction of the two major capsular polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GaIXM). These upstream steps are crucial for successful capsule construction, and therefore for cryptococcal virulence. Aim I focuses on a UDP-glucose dehydrogenase responsible for generating UDP-glucuronic acid, the activated donor of the glucuronic acid which is a major component of GXM. This protein has been expressed in active form, and efforts will focus on biochemical characterization, inhibitor studies, and gene disruption. Mutants defective in the activity will be generated and studied for their phenotype, virulence, and capsule structure. Aim II concerns a cryptococcal UDP-glucuronic acid decarboxylase discovered by the investigator and her coworkers, which is the first enzyme of its class to be cloned and expressed. This enzyme converts UDP-glucuronic acid to UDP-xylose, the donor of the xylose that is present in both major capsule glycans. C. neoformans cells disrupted in this gene demonstrate altered capsule structure and dramatically reduced virulence. Experiments will address the active site of the enzyme, enzyme inhibitors, and three dimensional structure. Aim III addresses the transport of nucleotide sugars into compartments in the cell where they serve as substrates for glycan synthesis. Initial studies will focus on a transporter cloned by the investigator's group. This protein will be characterized by in vitro assays and in vivo experiments, and a mutant with reduced expression of the protein will be tested for virulence and biochemically characterized. Other proteins identified by sequence analysis as putative transporters will be investigated, both by using RNA interference, recently shown by the investigator to effectively down-regulate gene expression in cryptococcus, and by biochemical studies. All of the processes under study are required for capsule synthesis, and are therefore essential for virulence of C. neoformans. Advances in understanding the biochemistry of nucleotide sugar synthesis and transport in this pathogen, and in particular progress in identifying inhibitors of the proteins involved, should contribute to the development of effective strategies for antifungal therapy.